1. Field of the Invention
The present invention relates to evaluating the permeability of the blood-neural barrier, and, more specifically, to evaluating the permeability of the blood-neural barrier by measuring PLA2 activity in cerebrospinal fluid.
2. Description of the Related Art
Barriers between the peripheral circulation and neural tissues are collectively referred to as blood-neural barriers (“BNB”) and include the blood-brain barrier (“BBB”) and the blood-cerebrospinal fluid barrier (“BCB”), among others. The BBB and the BCB play critical roles in the transport of substances into and out of the brain and cerebrospinal fluid (“CSF”), respectively. Their main functions are to protect the brain from potentially harmful substances in the blood such as cytokines and drug metabolites, to ensure a constant supply of nutrients, and to regulate brain-borne substances in order to maintain brain homeostasis. Due to its essential role in the maintenance of normal brain function, the integrity of the BNB is strictly regulated in part by tight junctions, localized between cerebral endothelial cells for the BBB and between choroid epithelial cells for the BCB, that form an almost impermeable barrier.
Disruption of the tight junction architecture or dysregulation of transporters can result in increased permeability of the BNB and can contribute to the pathophysiology of several brain disorders including neurodevelopmental diseases such as autism, schizophrenia, epilepsy and cerebral palsy; neurological lesions caused by ischemia or trauma; multiple sclerosis; and neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.
As a result, predictable and reliable ways to assess BNB permeability are useful for analyzing central nervous system disorders and to monitor time-dependent response to therapies that target BNB disruption. Moreover, associated with other measurements the BNB integrity evaluation might facilitate the differential diagnosis of neurodegenerative disorders or identify potential therapeutic windows to increase the accessibility and efficacy of therapeutic treatments.
A number of techniques have been created to assess the permeability of the BNB. For example, imaging approaches such as gadolinium enhancement in T1-weighted magnetic resonance imaging (MRI) scans are used to assess BBB impairment. This assessment, however, requires both intravenous administration of the potentially toxic compound gadolinium as well as highly specialized and expensive equipment and expertise. Other strategies take advantage of the fact that any disruption in the BNB integrity allows protein leakage in both directions and evaluation of permeability using either a blood-specific protein or a brain-specific protein such as albumin or S100β, respectively. The most common and well-established method to evaluate BNB permeability is the measurement of the ratio of serum albumin in CSF (“QAlb”). In normal adult individuals, the QAlb measurement is usually in the range of 6.8 to 10.2.
Despite the use of QAlb to evaluate BNB permeability, new methods are needed to increase reliability and reproducibility, and especially to increase assay sensitivity. With increased reproducibility and sensitivity, BNB permeability assays will allow researchers to, among other things, (i) identify neurologic disorders which are not currently associated with BNB impairment; (ii) increase the specificity of differential diagnosis between neurodegenerative disorders; (iii) monitor time-dependent response to therapies that target BNB disruption as well as disease progression; and (iv) identify potential therapeutic windows in order to increase drug efficacy.
A possible candidate protein for improved evaluation of BNB permeability is phospholipase. The phospholipase A2 (PLA2) family of isozymes catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids, resulting in the production of free fatty acids (e.g. arachidonic acid and docosahexaenoic acid) and lysophospholipids. The free fatty acids and lysophospholipids metabolites created by PLA2 enzymatic activity can serve as precursors for the synthesis of proinflammatory mediators such as eicosanoids (prostaglandins and leukotrienes) and platelet activating factor and can induce the expression of chemokines and cytokines. As a result, PLA2s are considered key inflammatory enzymes.
The secretory Ca2+-dependent phospholipases (sPLA2) are a member of the PLA2 family. The ten sPLA2 isozymes, which have been identified in many types of mammalian cells including the central nervous system, differ from other PLA2 enzymes by their low molecular weight, their requirement for millimolar calcium concentrations for catalytic activity, and their low selectivity for a specific phospholipid. Functionally, while the contribution of sPLA2 to various aspects of inflammation are well-documented, their involvement in BCB function has not been reported.